US10828377B2 - Method for determining presence or absence of suffering from malignant lymphoma or leukemia, and agent for treatment and/or prevention of leukemia - Google Patents
Method for determining presence or absence of suffering from malignant lymphoma or leukemia, and agent for treatment and/or prevention of leukemia Download PDFInfo
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- US10828377B2 US10828377B2 US15/770,947 US201615770947A US10828377B2 US 10828377 B2 US10828377 B2 US 10828377B2 US 201615770947 A US201615770947 A US 201615770947A US 10828377 B2 US10828377 B2 US 10828377B2
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Definitions
- the present invention relates to a method for determining whether a subject suffers from, or likely to suffer from malignant lymphoma or leukemia, an agent for treating and/or preventing malignant lymphoma or leukemia, and the like.
- Malignant lymphoma is a malignant neoplasm occurring in lymphoreticular tissue. Malignant lymphoma arises from malignant transformation of cells of the lymphoid lineage and is often found as a swollen lymph node, and infiltration into organs is also observed. Malignant lymphoma is a disease that often develops in elderly people, but also develops commonly in adolescents and young adults. The sites where lymphoma develops are lymph nodes, the spleen, and other sites where lymphoreticular cells locate. Malignant lymphoma is tentatively distinguished from lymphocytic leukemia by the site of occurrence and the tumorigenicity, but both diseases are biologically closely related and it may be difficult to clinically distinguish between the diseases.
- leukemia is a disease in which a hematopoietic cell in the process of differentiation becomes a tumor and clonally proliferates.
- Leukemia is classified roughly into acute leukemia, which progresses acutely with undifferentiated leukemia blast cells increase, and chronic leukemia, which progresses usually chronically with differentiated leukemia cells increase.
- acute leukemia in particular, acute lymphoblastic leukemia (ALL) is a disease that develops commonly, regardless of age. While childhood ALL has a relatively good prognosis, adolescent and young adult ALL or elderly ALL responds poorly to chemotherapy and there are almost no effective molecular targeted therapies (Non Patent Literatures 1 and 2).
- An object of the present invention is to provide a method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia and an agent for treating and/or preventing the disease.
- the present inventors have found that a fusion mutation of a DUX4 gene and an IGH or IGL gene, overexpression of a DUX4 gene, or a fusion mutation of an MEF2D gene specifically occurs in leukemia patients, by exhaustively analyzing sequences of cDNAs prepared from leukemia cells derived from leukemia patients with a next-generation sequencer. Furthermore, the present inventors have found that an inhibitor of DUX4 suppresses the proliferation of cell lines having the fusion mutation of a DUX4 gene and the overexpression of a DUX4 gene.
- the present invention is based on the foregoing findings and encompasses the following aspects.
- a method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia comprising:
- DUX4 gene encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 and
- the fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene is a mutation that forms a fusion gene comprising a part of the DUX4 gene on the 5′ terminal side and a part of the immunoglobulin heavy chain or immunoglobulin light chain gene on the 3′ terminal side.
- polypeptide encoded by the fusion gene of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene comprises:
- an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300 and lacking amino acids at positions 410 to 424 in the amino acid sequence set forth in SEQ ID NO: 2 on the N-terminal side; and 2 to 100 amino acids derived from an immunoglobulin heavy chain or immunoglobulin light chain gene on the C-terminal side;
- fusion mutation of an MEF2D gene is a mutation that forms a fusion gene comprising a part of the MEF2D gene on the 5′ terminal side and a part of the BCL9 gene on the 3′ terminal side;
- a polypeptide encoded by the fusion gene of an MEF2D gene and a BCL9 gene comprises:
- amino acid sequence comprising amino acids at positions 1 to 200 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426 in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side;
- fusion mutation of an MEF2D gene is a mutation that forms a fusion gene comprising a part of the MEF2D gene on the 5′ terminal side and a part of the HNRNPUL1 gene on the 3′ terminal side;
- a polypeptide encoded by the fusion gene of an MEF2D gene and an HNRNPUL1 gene comprises:
- amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side;
- a polypeptide comprising any of amino acid sequences of the following (i) to (iii):
- an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300 and lacking amino acids at positions 410 to 424 in the amino acid sequence set forth in SEQ ID NO: 2 encoded by a DUX4 gene on the N-terminal side; and 2 to 100 amino acids derived from an immunoglobulin heavy chain or immunoglobulin light chain gene on the C-terminal side;
- amino acid sequence of (i) comprises an amino acid sequence set forth in any of SEQ ID NOs: 10, 12, 14, 16, and 18.
- a marker for detecting malignant lymphoma or leukemia consisting of the polypeptide according to (8) or (9) described above or the polynucleotide according to (10) described above.
- a kit for detecting malignant lymphoma or leukemia comprising a primer set comprising a forward primer and a reverse primer for detecting a gene encoding DUX4 consisting of the amino acid sequence set forth in SEQ ID NO: 2 or an antibody that specifically binds to DUX4.
- An agent for detecting malignant lymphoma or leukemia comprising an antibody that specifically binds to DUX4.
- a pharmaceutical composition comprising a DUX4 inhibitor as an active ingredient, for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene and/or an overexpression of a DUX4 gene, wherein
- the DUX4 gene encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 and
- the fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene is a mutation that forms a fusion gene comprising a part of the DUX4 gene on the 5′ terminal side and a part of the immunoglobulin heavy chain or immunoglobulin light chain gene on the 3′ terminal side.
- the inhibitory nucleic acid is siRNA or shRNA.
- amino acid sequence comprising amino acids at positions 1 to 200 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426 in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side;
- amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side;
- a marker for detecting malignant lymphoma or leukemia consisting of the polypeptide according to any of (17) to (20) described above or the polynucleotide according to (21) described above.
- a kit for detecting malignant lymphoma or leukemia comprising a primer set comprising a forward primer and a reverse primer for specifically detecting the polynucleotide according to (21) described above and/or an antibody that specifically binds to the polypeptide according to any of (17) to (20) described above.
- a primer set comprising a forward primer and a reverse primer for specifically detecting a gene encoding a fusion protein of MEF2D and BCL9L, wherein
- the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in SEQ ID NO: 30 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in a sequence complementary to the sequence set forth in SEQ ID NO: 31; or
- the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of a sequence complementary to the sequence set forth in SEQ ID NO: 30 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 31 under stringent conditions.
- a primer set comprising a forward primer and a reverse primer for specifically detecting a gene encoding a fusion protein of MEF2D and HNRNPUL1, wherein
- the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in SEQ ID NO: 32 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in a sequence complementary to the sequence set forth in SEQ ID NO: 33; or
- the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of a sequence complementary to the sequence set forth in SEQ ID NO: 32 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 33 under stringent conditions.
- a method according to the present invention makes it possible to determine whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia rapidly and easily by detecting a novel marker gene or protein.
- the present invention may provide a pharmaceutical composition for effectively treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation or an overexpression of a DUX4 gene.
- FIG. 1 a is a pie graph showing the percentage of each gene fusion to all gene fusions detected in the screening cohort in Example 1. Gene fusions detected in ALLs of B-cell, T-cell, and unknown lineage are indicated with white, gray, and black colors, respectively.
- FIG. 1 b shows the frequency of DUX4- or MEF2D-fusion genes in the 3 generations (0 to 14 year old (children), 15 to 39 year old (AYA), 40 year old or older) in the combination cohort in Example 1.
- the screening for fusion genes was performed by RNA-seq in most of children or AYA, and RT-PCR was used in other examples.
- FIG. 1 c shows the Kaplan-Meier analysis of the screening cohort in Example 1.
- the patients were grouped into patients having Ph-like expression profiles, DUX4-fusion genes, MEF2D-fusion genes, or other non-Ph-like profiles.
- the P value was determined by the Logrank test.
- FIG. 2 a is a schematic view of an example of the DUX4 translocation in ALL.
- 2 copies of DUX4 having a fragment of mitochondrial genome were inserted between IGHJ6 and IGHV3-15 in the IGH locus.
- 1 copy of DUX4 was transferred with a fragment of chromosome 1 between IGHJ4 and IGHV3-66. In both reconfigurations, the 3′ terminal of DUX4 copies was disrupted.
- the symbol “Chr.” means chromosome.
- FIG. 2 b shows RNA-seq reads of Example 1 mapped onto the DUX4 locus and expressed as the number of reads per total 1,000,000 reads (RPM).
- the fusion points from DUX4 to IGH were indicated with arrows.
- the exon/intron structures of the DUX4 transcripts were illustrated below with the corresponding ID numbers (ENST numbers) in Ensemble transcript database.
- FIG. 2 c shows exemplary structures of fusion proteins of DUX4 and DUX4-IGH.
- the carboxy terminals of the DUX4-IGH fusion proteins found in the patients, # JALSG-001, # JALSG-003, # JALSG-004, or # JALSG-005 were fused with peptides encoded by genomic fragments of IGH. The number of amino acids is indicated under the schematic view of the structures of the proteins.
- FIG. 2 d shows the numbers of reads mapped onto DUX4 cDNA (NM_001293798) calculated from the RNA-seq data of the children and AYA cohorts in Example 1 and expressed as the numbers of mapped reads per total 1,000,000 RefSeq entries (RPM).
- Normal hemocytes three T cells and three CD19 + B-cell fractions in the peripheral blood and one CD10 + and CD34 + fraction in the bone marrow
- Ph + ALL specimens were analyzed similarly.
- the presence/absence of Ph or the DUX4 fusion gene is indicated below.
- the asterisk indicates the NALM6 cell line.
- FIG. 3 a shows the transformation ability of DUX4-IGH.
- 3T3 cells were transformed with DUX4 or DUX4-IGH genes identified in the patient # JALSG-001, # JALSG-003, # JALSG-004, or # JALSG-005 and these cells were subsequently cultured in 5% bovine serum for 12 days.
- Cells infected with retrovirus expressing no DUX4-IGH (Mock) were similarly analyzed.
- the scale bar is 100 ⁇ m.
- FIG. 3 b A shows that 2 copies of DUX4 were transferred to a position between IGHJ4 and IGHD2-15 in the genome of the NALM6 cell.
- B shows a protein encoded by DUX4-IGH fusion gene. In this protein, carboxy terminal of the DUX4 protein was substituted with 25 amino acids encoded by IGH.
- C shows the result of immunoblotting whole cell lysates of NALM6 transiently expressing DUX4-IGH or wild type DUX4 or HEK293 with an antibody to DUX4 or ACTB.
- FIG. 3 c NALM6 cells were infected with the recombinant retroviruses that express GFP with a control shRNA (control-sh) or DUX4-IGH shRNA (DUX4-sh) together with a virus that expresses DUX4-IGH identified in NALM6 or the patient # JALSG-003 (DUX4-sh+DUX-IGH (NALM6) or DUX4-sh+DUX-IGH (JALSG-003), respectively).
- the ratio of the GFP + fraction was tested by flow cytometry and standardized by the ratio on Day 3. The data are the mean ⁇ SD in a triplicate test.
- FIG. 4 a shows examples of MEF2D fusion detected in the cohort in Example 1.
- the number of amino acids is indicated under the schematic view of the structure of proteins.
- FIG. 4 b shows that an MED2D fusion is a gain of function mutation.
- Whole cell lysates prepared from HEK293 cells transfected with expression vectors of the wildtype MEF2D, MEF2D-BCL9, or MEF2D-HNRNPUL1 were subjected to the immunoblotting analysis with an antibody to MEF2D (upper panel) or ACTB (lower panel) (A).
- a reporter plasmid of MEF2D was transiently introduced into HEK293 cells with the control pGL-TK and a Mock plasmid or an expression vector of the wild type MEF2D, MEF2D-BCL9, or MEF2D-HNRNPUL1. The reporter activity was measured 2 days after transfection.
- the firefly luciferase activity was standardized with the Renilla luciferase activity. The value is mean ⁇ SD.
- FIG. 4 c shows the transformation ability of MEF2D fusion proteins.
- Murine 3T3 fibroblasts were infected with a Mock virus (Mock) or a recombinant retrovirus expressing the wild type MEF2D, MEF2D-BCL9, or MEF2D-HNRNPUL1, cultured in the presence of 5% bovine serum for 12 days and observed.
- the scale bar is 100 ⁇ m.
- FIG. 5 a shows the leukemogenesis activity of fusion oncogenes.
- Murine pro-B cells were infected with a Mock retrovirus or a recombinant virus expressing DUX4-IGH, MEF2D-HNRNPUL1, or MEF2D-BCL9 and transplanted into immunodeficiency mice exposed to radiation. 28 days after the transplant, expression of CD19, CD43, c-kit, CD25, IL7Ra, or IgM on the cell surface of B220 + GFP + cells among (primary) bone marrow cells was evaluated with a flow cytometer.
- the B220 + GFP + gating in the figure means that B220 + GFP + cells were used for the analysis and the abscissa axis indicates the expression intensity of B220 and the ordinate axis indicates the expression intensity of each protein indicated on the left edge.
- the same experiment was performed with secondary or tertiary transplant mice. ND means not determined.
- FIG. 5 b shows Kaplan Mayer analysis of the primary transplant mice in Example 3. The disease-free intervals of each group were compared by Logrank with Bonferroni correction.
- FIG. 5 c shows that the mice in which the DUX4-IGH expressing pro-B cells were transplanted develop leukemia.
- Cells were obtained from the bone marrow (BM), the spleen, or the liver in the aforementioned mice and tested for the expression of the differentiation marker proteins.
- the B220 + GFP + gating in the figure means that B220 + GFP + cells were used for the analysis and the abscissa axis indicates the expression intensity of B220 and the ordinate axis indicates the expression intensity of each protein indicated on the left edge.
- the lower right photograph shows the bone marrow smear-stained with the Wright-Giemsa solution.
- the scale bar is 5 ⁇ m.
- FIG. 6 is a schematic diagram of a gene in the specification.
- the gene in the genome is transcribed into mRNA and mRNA consisting of the coding region and the untranslated regions is produced by splicing.
- FIG. 7 shows an example when a polypeptide encoded by a fusion gene of a DUX4 gene and an IGH or IGL gene contains amino acids derived from an intron part of an IGH or IGL gene.
- the DUX4 gene was expressed as one region. The figure indicates that when DUX4 is inserted in an intronic part of an IGH or IGL gene and there is a stop codon in the intron part in the open reading frame that continues from DUX4 to the intron of the IGH or IGL, the polypeptide contains an amino acid sequence derived from a nucleotide sequence from a fusion part between DUX4 and the intron to the stop codon.
- FIG. 8 shows the result of immunostaining of a bone marrow cell sample derived from a leukemia patient using an anti-DUX4 polyclonal antibody.
- the present invention relates to a method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia, comprising: a detection step of detecting a fusion mutation of a DUX4 (Double homeobox 4) gene and an IGH (immunoglobulin heavy chain) or IGL (immunoglobulin light chain) gene and/or an overexpression of a DUX4 gene in a sample obtained from the subject; and a determination step of determining that the subject suffers from or is likely to suffer from malignant lymphoma or leukemia when the fusion mutation or the overexpression is detected.
- a detection step of detecting a fusion mutation of a DUX4 (Double homeobox 4) gene and an IGH (immunoglobulin heavy chain) or IGL (immunoglobulin light chain) gene and/or an overexpression of a DUX4 gene in a sample obtained from the subject
- the malignant lymphoma or leukemia is not limited to a particular kind.
- the malignant lymphoma include Hodgkin's lymphoma and non-Hodgkin's lymphoma and preferably a malignant lymphoma is B-cell lymphoma.
- the leukemia include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), etc.
- the leukemia is preferably ALL, particularly preferably child or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL).
- ALL particularly preferably child or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL).
- childhood means subjects aged 0 to 14 years and adolescent and young adult means subjects aged 15 to 39 years.
- the “subject” may be either of an individual that is suspected to suffering from malignant lymphoma or leukemia, or a normal individual.
- a method according to the present invention to an individual that is suspected to suffering from malignant lymphoma or leukemia, whether a subject suffers from malignant lymphoma or leukemia can be determined more accurately and, by applying a method according to the present invention to a normal individual, whether a subject suffers from malignant lymphoma or leukemia, or malignant lymphoma or leukemia, or the likelihood that the subject would suffer from malignant lymphoma or leukemia in the future can be determined.
- sample means a biological sample used in a method according to the present invention.
- the sample which can be used in the present invention includes, but not limited to, cells or tissues isolated from a living body.
- the cells include, for example, peripheral blood cells, lymph and tissue fluids containing cells, hair matrix cells, buccal cells, nasal cavity cells, intestinal tract cells, intravaginal cells, mucosal cells, and expectoration (that may contain alveolus cells or tracheal cells).
- the tissues include lesion sites, for example, the lymph node, the bone marrow, the spleen, the thymus, and the liver and, for example, biopsy samples of these tissues may be used.
- the “genes” encompass, unless otherwise specified, both coding regions that are transcribed into protein and structural genes (cistrons) encompassing coding regions plus untranslated regions that are not transcribed into protein and intron regions.
- FIG. 6 shows that a gene in the genome is transcribed into mRNA to produce mRNA consisting of coding regions and untranslated regions by splicing.
- the detection step of the method according to the present invention involves detecting a fusion mutation of a DUX4 gene and an IGH or IGL gene, preferably a fusion mutation of a DUX4 gene and an IGH gene, and/or an overexpression of a DUX4 gene.
- the fusion mutations and overexpression detected in the detection step of the method according to the present invention and detection step will be described in detail below.
- the “DUX4 gene” may be a gene encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 2.
- the DUX4 gene may be a gene encoding a polypeptide containing an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 2 by addition, deletion, and/or substitution of one or more amino acids.
- the value of the identity is expressed as a value calculated using a software (for example, FASTA, DANASYS, and BLAST) performing the operation for identity between a plurality of sequences with a default setting.
- a software for example, FASTA, DANASYS, and BLAST
- the range of “1 or more” is 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3 or 1 or 2.
- the gene encoding the amino acid sequence set forth in SEQ ID NO: 2 preferably contains the nucleotide sequence set forth in SEQ ID NO: 1.
- the “fusion mutation of a DUX4 gene and an IGH or IGL gene” means a fusion gene containing a part (preferably 5′ terminal side) of the DUX4 gene on the 5′ terminal side and a part of the IGH or IGL gene on the 3′ terminal side.
- the fusion mutation of a DUX4 gene and an IGH or IGL gene according to the present invention is generated preferably by the translocation of a partial region of the DUX4 gene to the IGH or IGL locus on genomic DNA as illustrated in FIG. 2 a .
- the fusion gene contains, from the 5′ terminal side to the 3′ terminal side, an IGH or IGL gene or a part thereof, a DUX4 gene or a part thereof, and an IGH or IGL gene or a part thereof.
- the polypeptide encoded by the fusion gene of a DUX4 gene and an IGH or IGL gene comprises preferably (i) an amino acid sequence comprising amino acids at positions 1 to 300, preferably positions 1 to 316, in the amino acid sequence set forth in SEQ ID NO: 2 and lacking amino acids at positions 410 to 424, preferably positions 409 to 424, on the N-terminal side and further comprising an IGH or IGL gene, preferably 2 to 100 amino acids, preferably 2 to 32 amino acids, derived from the IGH gene on the C-terminal side.
- the IGH or IGL gene is composed of a variety of sequences based on the gene rearrangement, the sequences thereof cannot be determined univocally.
- “comprising an amino acid sequence derived from an IGH or IGL gene” means comprising a sequence encoding a protein of the IGH or IGL gene; or a sequence derived from an intron part of the IGH or IGL gene.
- the fusion polypeptide “comprising a sequence derived from an intron part of the IGH or IGL gene” means, when the DUX4 gene or a part thereof is inserted in an intron part of the IGH or IGL gene and there is a stop codon in the intron part in an open reading frame following from DUX4 to the intron of IGH or IGL, the polypeptide comprising an amino acid sequence derived from a nucleotide sequence from a fusion part between DUX4 and the intron to the stop codon.
- FIG. 7 An example when a polypeptide encoded by a fusion gene of a DUX4 gene and an IGH or IGL gene contains amino acids derived from an intron part of an IGH or IGL gene is illustrated in FIG. 7 .
- Examples of the amino acid sequence of (i) include amino acid sequences set forth in any of SEQ ID NOs: 10, 12, 14, 16, and 18.
- the polypeptide encoded by the fusion gene may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above, or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the polypeptide encoded by the fusion gene has tumorigenic activity.
- the presence or absence of tumorigenic activity of a polypeptide can be specified by a known method in the art and determined, for example, by introducing a fusion gene into suitable cells (for example, mammalian cells), growing the cells, and observing the presence or absence of the focus formation.
- suitable cells for example, mammalian cells
- suitable cells for example, mammalian cells
- the gene fusion of DUX4 can be involved in the induction of malignant lymphoma or leukemia via at least one of the following three mechanisms: (i) increase of the DUX4 transcriptional activity caused by the translocation of DUX4 to the vicinity of the enhancer of IGH or IGL, (ii) reduction of apoptotic activity by deletion of the carboxy terminal of the encoded DUX4 protein, and (iii) translational activation of the DUX4 gene by a polyadenylation signal derived from IGH or IGL.
- the “overexpression” means expression that is statistically significantly higher than a reference.
- the reference include the amount of expression of the gene or protein obtained from a plurality (for example, 3 or more, 4 or more, or 5 or more) of normal subjects known not to suffer from malignant lymphoma or leukemia.
- the overexpression of DUX4 gene or protein means expression, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, for example, 10 times or more as high as the reference.
- the step of detecting a fusion mutation of a DUX4 gene and an IGH or IGL gene in the method according to the present invention comprises for example, a step of detecting the fusion gene in the genome, mRNA (or cDNA derived from mRNA) expressed from the fusion gene, or a polypeptide encoded by the fusion gene.
- the step of detecting overexpression of a DUX4 gene comprises, for example, a step of detecting mRNA (or cDNA derived therefrom) expressed from the gene or a polypeptide encoded by the gene.
- the detection of a gene in the genome may be performed using genomic DNA extracted, for example, by a known method.
- the extraction of genomic DNA may be performed using a commercially available DNA extraction kit.
- the step of detecting mRNA expressed from the fusion gene may be performed by using mRNA or by using cDNA reverse-transcribed from mRNA extracted from the sample.
- the extraction and the reverse transcription of mRNA may be performed by using, for example, commercially available mRNA extraction and reverse transcription kits.
- the step of detecting the fusion gene in the genome, or mRNA expressed from the fusion gene, or mRNA expressed from a DUX4 gene may be performed according to a known method for gene analysis (for example, PCR assay commonly used as a method for gene detection, RT-PCR, Real-time PCR, LCR (Ligase chain reaction), LAMP (Loop-mediated isothermal amplification), a microarray method, Northern hybridization, dot blotting, in situ hybridization, and an analysis by a next-generation sequencer, and the like).
- PCR assay commonly used as a method for gene detection
- RT-PCR Real-time PCR
- LCR Low-PCR
- LAMP Loop-mediated isothermal amplification
- a microarray method Northern hybridization, dot blotting, in situ hybridization, and an analysis by a next-generation sequencer, and the like.
- a gene amplification technique using a suitable primer or a hybridization technique with a suitable probe, or the like is used, using a nucleic acid extracted from a sample derived from a subject, for example, mRNA.
- the detection of a DNA or mRNA in the genome is performed qualitatively or quantitatively and, when performed quantitatively, the amount of DNA or mRNA may be measured, for example, by a method for the gene amplification reaction.
- the primer used for detecting the fusion gene or DUX4 gene in this detection step is not particularly limited, as long as they can be used to specifically amplify the fusion gene or DUX4 gene, and can be designed based on a nucleotide sequence of the fusion gene or DUX4 gene or mRNA expressed therefrom.
- the probe used for detecting the fusion gene or DUX4 gene in this detection step is not particularly limited, as long as they can specifically hybridize to the fusion gene or DUX4 gene, and can be designed based on a nucleotide sequence of the fusion gene or DUX4 gene or mRNA expressed therefrom.
- Examples of the primer and probe that can be used for detecting the fusion gene or DUX4 gene include the primer sets and probes described in “3. Primer, probe, and antibody for detecting mutation or overexpression of DUX4 gene” described below.
- the presence or absence of the fusion gene of a DUX4 gene and an IGH or IGL gene can be confirmed by a method suitable for each technology when the sequence of the fusion gene is known.
- the presence or absence of the mutation may be detected by analyzing the PCR product by agarose gel electrophoresis and confirming by ethidium bromide staining and the like whether an aimed size of amplification fragment has been obtained. For example, if an amplification fragment having a size corresponding to the fusion gene or mRNA expressed from the fusion gene is obtained, then it is presumed that the fusion gene is present in the sample obtained from the subject. As needed, the amplified fragment may be further sequenced.
- the presence or absence of the fusion gene can be detected.
- the presence or absence of the fusion gene of a DUX4 gene and an IGH or IGL gene can be specified by sequencing the terminal of the DUX4 gene by 3′-RACE in candidate specimens that may contain the fusion of a DUX4 gene and an IGH or IGL gene or exhaustive analysis of cDNAs from cells in the candidate specimens by a next-generation sequencer.
- the candidate specimens can be specified by, for example, the step of detecting overexpression of the fusion genes described below.
- the step of detecting a polypeptide encoded by the fusion gene may be performed by, for example, preparing a lysate derived from a sample obtained from a subject and conducting an immunological assay or an enzyme activity assay and the like for a polypeptide encoded by the fusion gene contained therein using an anti-DUX4 antibody or using an antibody to a polypeptide encoded by the fusion gene.
- an anti-DUX4 antibody or using an antibody to a polypeptide encoded by the fusion gene.
- techniques such as enzyme immunoassay, two-antibody sandwich ELISA, fluorescent immunoassay, radioimmunoassay, Western blotting, and the like using a monoclonal antibody or polyclonal antibody specific to a polypeptide encoded by the fusion gene may be used.
- the presence or absence of overexpression of the DUX4 gene can be confirmed by a method suitable for each technology.
- the overexpression of the DUX4 gene can be detected, for example, in Real-time PCR by amplifying a sample obtained from a subject with a fluorescence-labeled primer and comparing the fluorescence intensity with the value of a reference.
- the overexpression of DUX4 can be detected by, for example, immunostaining cells or tissue with an antibody to DUX4 and detecting the presence or absence of the fluorescence intensity or difference from a reference.
- the reference include samples obtained from a plurality (for example, 3 or more, 4 or more, or 5 or more) of normal subjects known not to suffer from malignant lymphoma or leukemia.
- the method according to the present invention may be used in combination with other methods (for example, X-ray radiography, endoscopy, and the like).
- other methods for example, X-ray radiography, endoscopy, and the like.
- the present invention relates to a polypeptide comprising (i) an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300, preferably positions 1 to 316, in the amino acid sequence set forth in SEQ ID NO: 2 coded by the DUX4 gene and lacking amino acids at positions 410 to 424, preferably positions 409 to 424 on the N-terminal side; and comprising 2 to 100 amino acids, preferably 2 to 32 amino acids, derived from an IGH or IGL gene on the C-terminal side.
- Examples of the amino acid sequence of (i) include amino acid sequences set forth in any of SEQ ID NOs: 10, 12, 14, 16, and 18.
- the present invention relates to a polypeptide comprising (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the polypeptide according to the present invention has tumorigenic activity.
- the presence or absence of the tumorigenic activity of the polypeptide can be specified by a known method in the art and determined, for example, by introducing a fusion gene into suitable cells (for example, mammalian cells), growing the cells, and observing the presence or absence of the focus formation.
- the polypeptide according to the present invention can be prepared from, for example, a sample derived from a subject determined to have the fusion gene or from a host cell into which a vector containing the fusion gene or fragment thereof is introduced.
- the purification of the peptide can be accomplished by performing a known method, for example, ammonium sulfate precipitation, separation by precipitation with an organic solvent (ethanol, methanol, acetone, etc.), chromatography such as ion exchange chromatography, isoelectric chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using a substrate, an antibody, or the like, reverse-phase column chromatography, HPLC, and the like, filtration treatment such as microfiltration, ultrafiltration, reverse osmosis, and the like alone or in combination.
- the present invention relates to a polynucleotide encoding a polypeptide according to the present invention.
- a polynucleotide include a polynucleotide comprising: (i) a nucleotide sequence set forth in any of SEQ ID NO: 9, 11, 13, 15, and 17; (ii) a nucleotide sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence of (i) described above; or (iii) a nucleotide sequence modified from the nucleotide sequence of (i) described above by addition, deletion, and/or substitution of one or more nucleotides.
- Examples of a method for preparing a polynucleotide according to the present invention include (1) a method using polymerase chain reaction (PCR); (2) a method involving selecting a transformant containing a desired polynucleotide from transformants transformed with a cDNA library; and (3) a method of chemical synthesis.
- PCR polymerase chain reaction
- a method for preparing a polynucleotide according to the present invention examples include (1) a method using polymerase chain reaction (PCR); (2) a method involving selecting a transformant containing a desired polynucleotide from transformants transformed with a cDNA library; and (3) a method of chemical synthesis.
- PCR polymerase chain reaction
- the present invention relates to a marker for detecting malignant lymphoma or leukemia, consisting of the polypeptide or polynucleotide.
- the present invention relates to a kit for detecting malignant lymphoma or leukemia.
- the kit according to the present invention comprises at least one of a primer set comprising a forward primer and a reverse primer for detecting a gene encoding DUX4 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a probe for detecting the gene, and an antibody that specifically binds to DUX4.
- the kit according to the present invention may comprise 2 or more of the at least one of the primer set, the probe, and the antibody.
- the primer set for detecting a gene encoding DUX4 is not particularly limited, as long as it can be used to specifically detect the DUX4 gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 29 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 29; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of a complementary sequence set forth in SEQ ID NO: 29 under stringent conditions and the reverse primer
- stringent conditions means conditions in which so-called specific hybrid is formed and no nonspecific hybrid is formed.
- the stringent conditions can be determined as appropriate, for example, with reference to Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press.
- stringent conditions may be set according to a temperature and a salt concentration contained in a solution in Southern hybridization and a temperature and a salt concentration contained in a solution in the washing step in Southern hybridization. More particularly, examples of the stringent conditions include a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68° C., preferably 42 to 65° C. More specifically, examples include 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) at a temperature of 42° C.
- the probe for detecting the gene encoding DUX4 is not particularly limited, as long as it can be used to detect the DUX4 gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 14, for example 20, preferably 30, consecutive nucleotide sequence in SEQ ID NO: 29 under stringent conditions, or (2) a polynucleotide that hybridizes to a polynucleotide consisting of a sequence complementary to at least 14, for example 20, preferably 30, consecutive nucleotide sequence in SEQ ID NO: 29 under stringent conditions.
- the primer and probe can be prepared by a known method known those skilled in the art, for example but not limited to, a method of chemical synthesis.
- the antibody that specifically binds to DUX4 may be a commercially available product or may be prepared by a method known to those skilled in the art.
- Examples of the antibody include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies (scFVs), humanized antibodies, fully human antibodies, and antibody fragments such as Fab, Fab′, F(ab′)2, Fc, Fv, and the like.
- Preferable examples include polyclonal or monoclonal antibodies.
- the antibody according to the present invention for example, a polyclonal antibody and a monoclonal antibody can be prepared using a target protein or a partial fragment thereof, for example, peptide containing SEQ ID NO: 41 or a cell expressing them, as a sensitized antigen in a method well known to those skilled in the art (E. Harlow et al. (Ed.), “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988).
- the polyclonal antibody can be obtained, for example, by administering an antigen such as a DUX4 protein or a fragment thereof and the like to a mammal such as a mouse or a rabbit, collecting blood from the mammal, and purifying the antibody.
- the method of immunization is well known to those skilled in the art and can be performed, for example, by administering an antigen one or more times.
- the antigen (or a partial fragment thereof) may be dissolved in a suitable buffer solution containing a commonly used adjuvant such as the complete Freund's adjuvant and used to increase the immunogenicity.
- the method for purifying an antibody is also known for those skilled in the art.
- the polyclonal antibody may be purified by conducting one or more of centrifugation, precipitation using ammonium sulfate and the like and chromatography, affinity chromatography using, for example, an antigen peptide, and the like.
- a method for producing a monoclonal antibody includes the hybridoma method.
- a mammal is immunized in a way similar to the production of a polyclonal antibody.
- the spleen is subsequently extracted from the immunized animal that has been sensitized to obtain B cells.
- the B cells were fused with myeloma cells according to a conventional method to generate antibody-producing hybridoma.
- any method known for those skilled in the art may be selected and used.
- the selection of the hybridoma may be performed by culturing cells for a suitable period of time in HAT medium (hypoxanthine, aminopterin, and thymidine-containing medium) according to a conventional method.
- HAT medium hyperxanthine, aminopterin, and thymidine-containing medium
- screening for and cloning of antibody-producing hybridoma of interest are conducted.
- the screening can be performed by a known method for detecting an antibody such as ELISA and the cloning may be performed by a method known to those skilled in the art such as limiting dilution.
- obtained hybridoma may be cultured in a suitable culture liquid and a monoclonal antibody can be purified from the culture liquid by salt precipitation, gel filtration, ion exchange chromatography, affinity chromatography, or the like.
- the primer set, probe, and antibody described above may have a label detectable by chemical or physical detection means to further facilitate the detection.
- the substance used as a label include fluorescent substances, enzymes, radioisotopes, luminescent substances, and the like.
- the fluorescent substances include fluorescamine, fluorescein isothiocyanate, and the like
- examples of the enzymes include peroxidase, alkaline phosphatase, and the like
- examples of the radioisotopes include 125 I, 131 I, 3 H, and the like
- examples of the luminescent substances include luciferin, luminol, derivatives thereof, and the like.
- the kit according to the present invention may comprise, for example, a buffer, an enzyme, an instruction, and the like, in addition to the primer set or probe described above.
- the present invention relates to an agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to DUX4.
- the agent for detecting malignant lymphoma or leukemia according to the present invention may be used as a reagent for sample analysis and the like or for the diagnosis of malignant lymphoma or leukemia.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a DUX4 inhibitor as an active ingredient, for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an IGH or IGL gene and/or an overexpression of a DUX4 gene.
- the inhibitor of DUX4 is not particularly limited, as long as it can inhibit the activity of DUX4, and examples thereof include inhibitory nucleic acids to DUX4, neutralization antibodies to DUX4, and low molecular weight compounds. These inhibitors may be used alone or in combination.
- inhibitory nucleic acids to DUX4 include nucleic acid aptamers to DUX4 and siRNAs or shRNA to DUX4, preferably siRNAs or shRNAs to DUX4.
- RNA small interference RNA
- shRNA short hairpin RNA refers to a single strand RNA in which the siRNA described above or a sense strand and an antisense strand of a mature double strand miRNA were connected with a short spacer sequence having a suitable sequence.
- shRNA forms a hairpin-shaped stem-loop structure as a whole molecule by a sense region and an antisense region forming base-pairing between each other in a molecule to form a stem structure, and a spacer sequence simultaneously forming a loop structure.
- the inhibitory nucleic acid is preferably introduced into a vector and administered to a subject and examples of vectors that can be used for the delivery of the inhibitory nucleic acid include the pLMN vector (Transomic).
- the pharmaceutical composition according to the present invention may comprise an active ingredient, for example, an anticancer agent, such as 5-FU (fluorouracil), methotrexate, leucovorin, trastuzumab, and the like, other than the inhibitor of DUX4.
- an anticancer agent such as 5-FU (fluorouracil), methotrexate, leucovorin, trastuzumab, and the like, other than the inhibitor of DUX4.
- the pharmaceutical composition according to the present invention can be formulated by a method known in the field as a general rule. For example, it can be formulated using a method described in Remington's Pharmaceutical Sciences (Merck Publishing Co. Easton, Pa.). Specific methods of formulation vary depending on the mode of administration. Modes of the administration are roughly classified into the oral administration and the parenteral administration and the mode of administration can be selected as appropriate.
- a pharmaceutically acceptable carrier may be added.
- the “pharmaceutically acceptable carrier” refers to a substance that facilitates the formulation of an agent and the application to the living body is added in a range that does not inhibit or suppress the effect of the active ingredient. Examples thereof include excipients, binders, disintegrators, fillers, emulsifiers, flow additives and modifiers or lubricants.
- excipients include sugar such as monosaccharides, disaccharides, cyclodextrin, and polysaccharides (specifically including, but are not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (for example, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate), citric acid, tartaric acid, glycine, low, medium, high molecular weight polyethyleneglycol (PEG), Pluronic, or a combination thereof.
- sugar such as monosaccharides, disaccharides, cyclodextrin, and polysaccharides (specifically including, but are not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin,
- binder examples include starch paste using starch of corn, wheat, rice, or potato, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone and the like.
- disintegrators examples include the starch or carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, or a salt thereof.
- fillers examples include the sugar and/or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).
- emulsifiers examples include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.
- flow additives and modifiers examples include silicates, talc, stearates, or polyethyleneglycol.
- Examples of the dosage form of an oral formulation include solid preparations (including tablets, pills, lingusorbs, capsules, and drops), granules, powdered medicines, powders, solutions, and the like.
- the solid preparations can be formulated into a dosage form with a coating known in the field, for example, sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, double-coated tablets, multilayered tablets, as needed.
- a coating known in the field, for example, sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, double-coated tablets, multilayered tablets, as needed.
- Either of the specific shape and size of the dosage form is not particularly limited as long as it is in the range of dosage forms known in the field for each dosage form.
- the pharmaceutical composition according to the present invention When the pharmaceutical composition according to the present invention is parenterally administered, specific examples thereof include administration by injection.
- the pharmaceutical composition according to the present invention When the pharmaceutical composition according to the present invention is administered by injection, the pharmaceutical composition may be prepared as a suspension in which the inhibitor is mixed with a pharmaceutically acceptable solvent and a pharmaceutically acceptable carrier was added to the mixture, as needed.
- the “pharmaceutically acceptable solvent” may be any of water or another pharmaceutically acceptable aqueous solution or an oily liquid.
- aqueous solution include physiological saline and isotonic solutions containing glucose or another pharmaceutic aid.
- pharmaceutic aid include D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and low-concentrations of non-ionic surfactants (for example, polysorbate 80 (TM), HCO-60), polyoxyethylene sorbitan fatty acid esters, and the like.
- Example of the oily liquid include sesame oil and soybean oil and may be used in combination with for example, benzyl benzoate or benzyl alcohol as a solubilizing agent.
- a buffer for example, a phosphate buffer solution, a sodium acetate buffer solution, a soothing agent, for example, hydrochloric acid procaine, a stabilizer, for example, benzyl alcohol, phenol, an antioxidant may be blended.
- the injections may be formulated by mixing the inhibitor in combination with a pharmaceutically acceptable excipient, emulsifier, suspension, surfactant, stabilizer, pH regulator, or the like, as appropriate in a unit dose form required in generally accepted pharmaceutical practices.
- injection examples include intravascular injection, intralymphatic injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like and injection is preferably cardiovascular administration such as intravascular injection or intralymphatic injection, which is systemic administration, but may be a topical administration involving the direct administration into lymphoma.
- the content of the active ingredient in the pharmaceutical composition according to the present invention may be an amount that may result in the delivery of the active ingredient to the target site by one time of administration in principle and that has almost or completely no adverse side effects to the subject to which the composition is administered.
- Such content varies depending on the stage of the disease, the kind of the DUX4 inhibitor, the dosage form of the pharmaceutical composition, the mode of administration, and the like, but is determined as appropriate by a person skilled in the art.
- fusion mutations of the DUX4 gene and overexpression of the DUX4 gene that a subject to which the pharmaceutical composition according to the present invention is administered (subject to which the pharmaceutical composition according to the present invention is to be applied) has are as described in “1.
- Examples of the kind of malignant lymphoma and leukemia to be treated and/or prevented with the pharmaceutical composition according to the present invention include the aforementioned Hodgkin's lymphoma and non-Hodgkin's lymphoma for malignant lymphoma and the malignant lymphoma is preferably B-cell lymphoma.
- leukemia examples thereof for leukemia include the aforementioned acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia and the leukemia is preferably ALL, particularly preferably childhood or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL).
- ALL particularly preferably childhood or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL).
- the pharmaceutical composition according to the present invention may be administered to, for example, a subject to which the method according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied.
- Administration of the pharmaceutical composition according to the present invention to a subject suffering from malignant lymphoma or leukemia is expected to have a therapeutic effect and administration of the pharmaceutical composition according to the present invention to a subject who is likely to suffer from malignant lymphoma or leukemia is expected to have a preventive effect.
- the present invention relates to a method for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an IGH or IGL gene and/or an overexpression of a DUX4 gene, the method comprising administering the aforementioned DUX4 inhibitor or the aforementioned pharmaceutical composition as an active ingredient.
- a method for treatment and/or prevention according to the present invention may preferably be performed to a subject to which the method for determination according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied. That is, administration of the DUX4 inhibitor or the aforementioned pharmaceutical composition to a subject determined to suffer from malignant lymphoma or leukemia by the aforementioned method for determination is expected to have a therapeutic effect and administration of the DUX4 inhibitor or the aforementioned pharmaceutical composition to a subject determined to be likely to suffer from malignant lymphoma or leukemia is expected to have a preventive effect.
- the kind of malignant lymphoma and leukemia to be treated and/or prevented by the method according to the present invention is as described above.
- the present invention relates to a method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia, comprising: a detection step of detecting a fusion mutation of an MEF2D (Monocyte-specific enhancer factor 2) gene in a sample obtained from a subject, and a determination step of determining that the subject suffers from or is likely to suffer from malignant lymphoma or leukemia when the fusion mutation is detected.
- MEF2D Monocyte-specific enhancer factor 2
- the “MEF2D gene” may be a gene encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 4.
- the MEF2D gene may be a gene encoding a polypeptide containing an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 4 by addition, deletion, and/or substitution of one or more amino acids.
- the gene encoding the amino acid sequence set forth in SEQ ID NO: 4 preferably contains the nucleotide sequence set forth in SEQ ID NO: 3.
- the fusion mutations of an MEF2D gene are fusion mutations of an MEF2D gene and a BCL9 gene or an MEF2D gene and an HNRNPUL1 gene.
- the “fusion mutations of an MEF2D gene and a BCL9 gene” mean fusion genes containing a part of the MEF2D gene (preferably 5′ terminal side) on the 5′ terminal side and a part of the BCL9 (B-cell Lymphoma 9) gene (preferably 3′ terminal side) on the 3′ terminal side.
- the polypeptides encoded by the fusion genes comprise (i) an amino acid sequence comprising amino acids at positions 1 to 200, preferably positions 1 to 202, in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426, preferably positions 1055 to 1426, in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side.
- Examples of the amino acid sequence of (i) include any of SEQ ID NOs: 20, 22, 24, and 26, preferably the amino acid sequence set forth in SEQ ID NO: 20 or 22.
- polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the “fusion mutations of an MEF2D gene and an HNRNPUL1 gene” mean fusion genes containing a part of the MEF2D gene (preferably 5′ terminal side) on the 5′ terminal side and a part of the HNRNPUL1 (Heterogeneous Nuclear Ribonucleoprotein U-Like 1) gene (preferably 3′ terminal side) on the 3′ terminal side.
- the polypeptides encoded by the fusion genes comprise (i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side.
- amino acid sequence of (i) examples include the amino acid sequence set forth in any of SEQ ID NO: 28.
- the polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the polypeptides encoded by the MEF2D fusion genes have tumorigenic activity. Methods for determining the presence or absence of the tumorigenic activity of the polypeptide and a detection step of detecting a fusion mutation of a MEF2D gene are as described in “1. Method for determining disease based on mutation or overexpression of DUX4 gene” described above and therefore not described here.
- the present invention relates to a fusion polypeptide of MEF2D and BCL9.
- the fusion polypeptides comprise (i) an amino acid sequence comprising amino acids at positions 1 to 200, preferably positions 1 to 202, in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426, preferably positions 1055 to 1426, in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side.
- Examples of the amino acid sequence of (i) include any of SEQ ID NOs: 20, 22, 24, and 26, preferably the amino acid sequence set forth in SEQ ID NO: 20 or 22.
- polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the present invention relates to a fusion polypeptide of MEF2D and HNRNPUL1.
- the fusion polypeptide comprises (i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side.
- Examples of the amino acid sequence of (i) include the amino acid sequence set forth in any of SEQ ID NO: 28.
- polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
- the fusion polypeptide has tumorigenic activity. Methods for measuring the presence or absence of the tumorigenic activity of the polypeptide are as described in “1. Method for determining disease based on mutation or overexpression of DUX4 gene” described above and therefore not described here.
- the present invention relates to a polynucleotide encoding the fusion polypeptide.
- a polynucleotide include a polynucleotide comprising: (i) a nucleotide sequence set forth in any of SEQ ID NOs: 19, 21, 23, 25, and 27; (ii) a nucleotide sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence of (i) described above; or (iii) a nucleotide sequence modified from the nucleotide sequence of (i) described above by addition, deletion, and/or substitution of one or more nucleotides.
- Methods for preparing the fusion polypeptide and polynucleotide are similar to the method described in “2. DUX4 fusion polypeptide and polynucleotide” and therefore not described here.
- the present invention relates to a marker for detecting malignant lymphoma or leukemia consisting of the polypeptide or polynucleotide.
- an MEF2D fusion polypeptide or polynucleotide can be used as a prognosis marker. Since it is expected that prognosis would be bad, for example, when a fusion mutation of an MEF2D gene is detected, the likelihood of recurrence of malignant lymphoma or leukemia can be reduced by conducting treating or preventing measures such as bone marrow transplantation beforehand.
- the present invention relates to a primer set comprising a forward primer and a reverse primer for detecting a fusion gene of an MEF2D gene and a BCL9 gene.
- the primer set is not particularly limited, as long as it can be used to specifically detect the fusion gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 30 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 31; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes
- the present invention relates to a probe for detecting a fusion gene of an MEF2D gene and a BCL9 gene.
- the probe is not particularly limited, as long as it can be used to detect the fusion gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 30 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 31 under stringent conditions; or (2) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 30 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 31 under stringent conditions.
- the present invention relates to a primer set comprising a forward primer and a reverse primer for detecting a fusion gene of an MEF2D gene and an HNRNPUL1 gene.
- the primer set is not particularly limited, as long as it can be used to specifically detect the fusion gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 32 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 33; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridize
- the present invention relates to a probe for detecting a fusion gene of an MEF2D gene and an HNRNPUL1 gene.
- the probe is not particularly limited, as long as it can be used to detect the fusion gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 32 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 33 under stringent conditions; or (2) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 32 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 33 under stringent conditions.
- the primer and probe can be prepared by a method known to those skilled in the art, for example but not limited to, by a method of chemical synthesis.
- the present invention relates to an antibody that specifically binds to a fusion protein of MEF2D and BCL9 or a fusion protein of MEF2D and HNRNPUL1.
- the kind of and the method for preparing the antibody are similar to those described in “3. Primers, probes, and antibodies for detecting mutation or overexpression of DUX4 gene” described above and therefore not described here.
- the antibody to the aforementioned fusion protein may be prepared by a method known to those skilled in the art.
- a mammal is immunized with a protein containing a fusion part and an antibody can be obtained from the serum by any method known to those skilled in the art.
- the primer set, probe, and antibody described above may have a label detectable by chemical or physical detection means to further facilitate the detection.
- the substance used as a label include fluorescent substances, enzymes, radioisotopes, luminescent substances, and the like.
- the fluorescent substances include fluorescamine, fluorescein isothiocyanate, and the like
- examples of the enzymes include peroxidase, alkaline phosphatase, and the like
- examples of the radioisotopes include 125 I, 131 I, 3 H, and the like
- examples of the luminescent substances include luciferin, luminol, derivatives thereof, and the like.
- the present invention relates to a kit for detecting malignant lymphoma or leukemia comprising at least one of the aforementioned primer set, probe, and antibody.
- the kit according to the present invention may comprise, for example, a buffer, an enzyme, an instruction, and the like, in addition to the probe or antibody.
- the present invention relates to an agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to a fusion protein of MEF2D and BCL9 or a fusion protein of MEF2D and HNRNPUL1.
- the agent for detecting malignant lymphoma or leukemia according to the present invention may be used as a reagent for sample analysis and the like or for the diagnosis of malignant lymphoma or leukemia.
- the present invention relates to a pharmaceutical composition for treating and/or preventing malignant lymphoma or leukemia, comprising an MEF2D inhibitor as an active ingredient in a subject having the aforementioned fusion mutation of the MEF2D gene.
- the MEF2D inhibitor is not particularly limited, as long as it can inhibit the activity of MEF2D, and examples thereof include inhibitory nucleic acids to MEF2D, neutralizing antibodies to MEF2D, and low molecular weight compounds. These inhibitors may be used alone or in combination.
- composition comprising inhibitor of DUX4” described above and therefore not described here.
- the present invention relates to a method for treating and/or preventing malignant lymphoma or leukemia in a subject having the aforementioned fusion mutation of the MEF2D gene, comprising administering the aforementioned MEF2D inhibitor or the aforementioned pharmaceutical composition to the subject.
- a method for treatment and/or prevention according to the present invention may preferably be performed to a subject to which the method for determination according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied.
- HEK 293 cells and murine 3T3 fibroblasts were obtained from American type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) (Thermofisher) containing 10% fetal bovine serum (FBS) (Thermofisher).
- DEM-F12 Dulbecco's modified Eagle's medium-F12
- FBS fetal bovine serum
- B-cell lines Kasumi-7, Kasumi-9, NALM6, NAGL-1, and CCRF-SB
- RPMI1640 medium Thermofisher
- CD19 + B-cell and T-cell fractions were selected from the peripheral blood of normal volunteers by a magnetic beads-purification system (Miltenyi Biotech). Bone marrow CD34 + cells were purchased from Takara Bio. The CD10+ fraction was concentrated from bone marrow mononuclear cells purchased from Takara Bio by a magnetic beads purification system.
- the validation cohort consisted of 62 patients with B-cell ALL including 5 recurrences. The median age was 24 year old (in a range of 0 to 81 year old). The samples of complete remission were obtained from 34 patients. In addition, this study has obtained informed consents from all patients and been approved by the Ethical Review Board of all participated research institutes including University of Tokyo.
- RNA was prepared from RNA using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) and subjected to NGS sequencing from the both ends in HiSeq2000/2500 platform (Illumina).
- RNA-seq data the known fusion genes exhibited the following parameter values in the deFuse analysis; Probability >0.9 and split read count >100. Therefore, Round 1 of screening for fusion genes was conducted with the same parameters as those that give yes for the inframe option (Algorithm A). Subsequently, in the next round of screening, fusion genes were searched for using the probe genes specified with Algorithm A as described above (set forth in the Table below) or already known to be involved in gene fusions, by the following standards; Probability >0.9 or split read count >100 (Algorithm B).
- the fusion gene candidates specified with Algorithm A or Algorithm B were confirmed by RT-PCR and subsequent sequencing.
- RNA-seq data For expression profiling by RNA-seq data, paired-end reads were aligned to hg19 human genome assembly using TopHat2. The expression levels of respective RefSeq genes were calculated from read counts mapped using HTSeq and standardized by DESeq2 pipeline. For the clustering analysis, the standardized read counts were further converted by dispersion visualization conversion in DESeq2 and subjected to the hierarchical clustering analysis by the Ward's method. The genes used for the clustering have been defined in Roberts et al., N. Engl. J. Med., 371, pp. 1005-1015 (2014), but slightly modified according to the recent Refseq database.
- sequence reads with Q value for each base ⁇ 20 were selected from the data set and mapped onto the RNAseq database using the Bowtie2 algorithm. Mismatches were removed when (i) the obtained read contains ⁇ 3 independent mismatches; (ii) they are already listed as a genome change in normal humans in the “1000 genomes” database or the in-house database of the present inventors; or (iii) they are supported by only one strand in the genome. Genetic mutations were annotated by SnpEff.
- Genomic DNA was subjected to PCR amplification using the following primers:
- DUX4-IGH (SEQ ID NO: 34) 5′-ATAACGGTGTCCTTCTGTTTGCAG-3′ and (SEQ ID NO: 35) 5′-GCAGAGGGGATCTCCCAACCT-3′; MEF2D-BCL9: (SEQ ID NO: 36) 5′-CAGCCAGCACTACAGAGGAACAG-3′ and (SEQ ID NO: 37) 5′-GGCATCTGATTGGAGTGAGAAAGT-3′; and ACTB: (SEQ ID NO: 38) 5′-CTTCTCCTTAATGTCACGCACGAT-3′ and (SEQ ID NO: 39) 5′-GATCATGTTTGAGACCTTCAACACC-3′.
- TCF3-PBX1 was identified in 4 individuals, STIL-TAL1 in 4 individuals, ETV6-RUNX1 in 1 individual, KMT2A-MLLT1 in 1 individual, and KMT2A-AFF3 in 1 individual (STIL-TAL1 was identified in T-cell ALL and the others were identified in B-cell ALL).
- Fusion gene candidates were searched for from the data set using a computer pipeline by the deFuse algorithm (McPherson, A. et al., PLoS Comput. Biol., 7, e1001138 (2011)).
- the fused transcription products which were already identified in PCR were successfully detected in correspondent specimens.
- 26 independent fusion genes in total were found and 11 of them were novel ( FIG. 1 a ).
- No fusion candidates were identified in 14 patients with B-cell AYA-ALL (25.9%) or 10 patients (55.6%) with T-cell AYA-ALL.
- B-cell ALL can be classified into Ph-like or non-Ph-like and the tyrosine kinase pathway is often activated in Ph-like B-cell ALL.
- the clustering analysis with no objective variable of the screening cohort including cases of Ph-positive ALL was conducted using RNA-seq data and it was revealed that 7 patients with Ph-negative ALL as well as 3 patients with Ph-positive ALL were classified into Ph-like ALL.
- the clinical result (data not shown) of genetic mutation profiling of the screening cohort has indicated that the gene fusions are mutually exclusive.
- the DUX4- and MEF2D-fusions were all classified into the non-Ph-like group. However, the effects of these mutations on prognosis of patients are different.
- the D4Z4 repeat in which the DUX4 gene locates, was found to be mainly translocated to the IGH locus in chromosome 14 in the AYA-ALL subset ( FIG. 2 a ). Since the NGS data analysis in the study of the present inventors was an analysis with a mean sequence read length of 104 nucleotides, it was difficult to determine the copy number of DUX4 inserted into the IGH locus. However, the sudden decrease in the number of reads mapped on the DUX4 locus across the fusion point as illustrated in FIG. 2 b suggests that several copies of the DUX4 gene were translocated.
- RACE rapid amplification of cDNA ends
- the MEF2 reporter plasmid was purchased from Qiagen.
- a human MMP7 promoter region (Corveleyn, A. et al., J. Cell. Biochem. 94, 1112-1125 (2005)) was amplified by PCR from human genomic DNA and ligated to the luciferase vector pGL3 (Promega). 100 ng of the reporter plasmid, 200 ng of the expression vector, and 4 ng of pGL-TK (Promega) were transfected into HEK293T cells using Lipofectamine LTX reagent (Thermofisher). 48 hours later, the cells were lysed and the luciferase activity was measured. The reporter activity based on the firefly luciferase was standardized with the activity of Renilla luciferase.
- recombinant retroviruses were produced by introducing a pMXS- or pMSCV-based expression vector and ecotropic packaging plasmid (Takara Bio) into HEK293T cells. 3T3 cells were infected with the recombinant retroviruses and the cells were then cultured for 2 weeks in DMEM-F12 (Thermofisher) supplemented with 5% bovine serum (Thermofisher).
- Expression vectors for various cDNAs were transiently transduced into HEK293T cells using Lipofectamine LTX and the cells were lysed 2 days later.
- the immunoblot analysis was performed using antibodies to DUX4 (ab124699) or MEF2D (ab93257) (both from Abcam).
- the cell lysates were also analyzed by immunoblotting with an antibody to ACTB (#4967, Cell Signaling Technology).
- a shRNA expression vector was constructed using the vector pLMN (Transomic).
- the sequence of shRNA to DUX4-IGH is 5′-ACCCUGUGUGUCUCAGUUCAUA-3′ (SEQ ID NO: 40) and this targets an IGH region in the transcript of a DUX4-IGH fusion gene in NALM6.
- the wildtype DUX4 or DUX4 fusion genes were expressed in murine 3T3 fibroblasts and the focus formation assay was performed. As illustrated in FIG. 3 a , all the fibroblasts into which DUX4-fusion genes were introduced were transformed to exhibit the oncogenic activity, but the fibroblasts into which the wild type DUX4 was introduced did not exhibit the oncogenic activity.
- RNA-seq of total 5 cell lines with B-cell ALL revealed high expression of DUX4 in NALM6 established from a male aged 19 years ( FIG. 2 d ).
- Screening for fusion genes confirmed that the NALM6 cell line actually had DUX4-IGH ( FIG. 3 b ).
- the fusion with IGH resulted in a DUX4 protein having a mutant carboxy terminal and the expression thereof was confirmed by the immunoblotting analysis. This rearrangement locates the polyadenylation signal in IGHD2-15 in the vicinity of the DUX4 protein-coding region.
- MEF2D-BCL9 and MEF2D-HNRNPUL1 were found in 2 individuals and 3 individuals respectively in the AYA-ALL cohort of the present invention ( FIG. 4 a ). Moreover, it was proved that MEF2D-BCL9 fusion genes derived from the rearrangement of the somatic cell genome (data not shown).
- Both MEF2D-BCL9 and MEF2D-HNRNPUL1 proteins had high transcriptional activity relative to the corresponding wild type protein ( FIG. 4 b ).
- the cells expressing the fusion proteins exhibited a plurality of abnormal focuses, but no abnormal focuses were found with those expressing the wildtype protein ( FIG. 4 c ).
- B220+c-kit+pro-B cells were induced from fetus hepatocytes cultured on OP9 cells in Iscove's modification Dulbecco medium (Thermofisher) supplemented with 15% FBS, Stem cell factor, flt3 ligand, interleukin-7, and 2-mercaptoethanol.
- GFP-positive cells were inoculated with NSG mice (Jackson Laboratory) exposed to a sublethal dose (2 grays) of radiation. 28 days after the transplant, the expression of CD19, CD43, c-kit, CD25, IL7Ra, or IgM on the cell surface of B220 + GFP + cells among the (primary) bone marrow cells was evaluated with a flow cytometer.
- anti-B220 (RA3-6B2), anti-CD19 (1d3), anti-c-kit (2B8), anti-CD43 (S7), anti-CD25 (PC61.5), and anti-IgM (II/41) antibodies were used. All antibodies except S7, which was purchased from BD Biosciences, were obtained from eBioscience. All animal experiments were conducted following the approved protocols by Institutional Animal Care and Use Committee at Aichi Cancer Center and Nagoya University.
- DUX4-IGH fusion cDNA was introduced into murine pro-B cells with retrovirus and the cells were injected into immunodeficiency mice.
- the DUX4-IGH expression pro-B cells were proliferated in vivo, but maintained at an immature stage (CD19+CD43+c-kit+ CD25 ⁇ IL7Ra+ IgM ⁇ ) ( FIG. 5 a , FIG. 5 c ).
- These cells can be transplanted successively into secondary and tertiary recipients, which indicates that they have self-renewal activity.
- the wildtype DUX4 is introduced using the same retrovirus vector, the murine pro-B cells caused apoptosis.
- the murine transplant assay was conducted again with sorted B cells that were infected with a modified recombinant retrovirus to express a small amount of DUX4 under control of an internal ribosome entry site (ires) fragment.
- ires internal ribosome entry site
- MEF2D-BCL9-expressing B cells were grown in vivo using the same transplant assay, MEF2D-BCL9 does not provide the in vivo proliferation superiority to the pro-B cells, but both MEF2D-BCL9 and MEF2D-HNRNPUL1 prevented the differentiation of the B cell at the stage of the pro-B cell in 4 of 17 mice ( FIG. 5 a ).
- mice finally developed leukemia with a median incubation period of 157 days ( FIG. 5 b ).
- MEF2D-BCL9 has a lower penetrance, but also induces leukemia (up to 50% in an observation period of 290 days).
- Anti-DUX antibodies were produced using 1 to 13 amino acid residues (MALPTPSDSTLPA: SEQ ID NO: 41) of the human DUX4 protein according to a conventional method in a rabbit and an anti-DUX4 polyclonal antibody was affinity purified with the aforementioned peptide. Subsequently, the obtained antibody (1.2 ⁇ g/ml) was contacted with bone marrow cells derived from leukemia patients for 32 minutes at room temperature and then detection was made by BenchMark XT system (Ventana Medical Systems Inc) according to instructions of the manufacturer.
- BenchMark XT system Ventana Medical Systems Inc
Abstract
Description
- Non Patent Literature 1: Pulte, D., Gondos, et al., Blood, 2009, 113, pp. 1408-1411.
- Non Patent Literature 2: Schafer, E. S. et al., Nat. Rev. Clin. Oncol., 2011, 8, pp. 417-424.
(13) An agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to DUX4.
(14) A pharmaceutical composition comprising a DUX4 inhibitor as an active ingredient, for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene and/or an overexpression of a DUX4 gene, wherein
(16) The pharmaceutical composition according to (15) described above, wherein the inhibitory nucleic acid is siRNA or shRNA.
(17) A polypeptide comprising any of amino acid sequences of the following (i) to (iii):
(24) A primer set comprising a forward primer and a reverse primer for specifically detecting a gene encoding a fusion protein of MEF2D and BCL9L, wherein
TABLE 1 | ||||||
IGHJ1-6 | PAX5 | ZNF384 | NUP98 | BCL2 | ||
IGHM | RUNX1 | DUX4 | CRLF2 | |||
TRA | PBX1 | MYC | PDGFRB | |||
TRB | CEBP family | TAL1 | JAK2 | |||
TRD | MEF2D | TLX3 | KMT2A | |||
DUX4-IGH: | |
(SEQ ID NO: 34) | |
5′-ATAACGGTGTCCTTCTGTTTGCAG-3′ | |
and | |
(SEQ ID NO: 35) | |
5′-GCAGAGGGGATCTCCCAACCT-3′; | |
MEF2D-BCL9: | |
(SEQ ID NO: 36) | |
5′-CAGCCAGCACTACAGAGGAACAG-3′ | |
and | |
(SEQ ID NO: 37) | |
5′-GGCATCTGATTGGAGTGAGAAAGT-3′; | |
and | |
ACTB: | |
(SEQ ID NO: 38) | |
5′-CTTCTCCTTAATGTCACGCACGAT-3′ | |
and | |
(SEQ ID NO: 39) | |
5′-GATCATGTTTGAGACCTTCAACACC-3′. |
<Result>
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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EP2426203A2 (en) | 2010-09-02 | 2012-03-07 | Université de Mons | Agents useful in treating facioscapulohumeral muscular dystrophy |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005082933A1 (en) | 2004-02-26 | 2005-09-09 | Bml, Inc. | Novel chimeric protein, gene encoding the same, and means of judging leukemia using the gene and the protein |
EP2426203A2 (en) | 2010-09-02 | 2012-03-07 | Université de Mons | Agents useful in treating facioscapulohumeral muscular dystrophy |
US20120225034A1 (en) | 2010-09-02 | 2012-09-06 | Universite De Mons | Agents useful in treating facioscapulohumeral muscular dystrophy |
US20140105873A1 (en) | 2010-09-02 | 2014-04-17 | Universite De Mons | Agents useful in treating facioscapulohumeral muscular dystrophy |
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